Effects of Glucagon-like Peptide-1-(7-36)amide (GLP-1)
on Gastric Motor Function in Health and Diabetes:
Potential Mechanism of Action
Doctoral thesis submitted to the Autonomous University of
Barcelona by
Sílvia Delgado-Aros, M.D.
Director: Michael Camilleri M.D., Professor of Medicine and Physiology. Mayo Clinic Rochester and Mayo Foundation
Tutor: Juan Ramón Malagelada Benaprès M.D., Professor of
Medicine. Autonomous University of Barcelona
October 2002
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Table of Contents
Page Number
1. General Introduction 3
Introductory Schema 6
2. Studies
2.1 Effect of GLP-1 on Gastric Volume, Emptying, Maximum Volume
Ingested, and Postprandial Symptoms in Humans
2.1.1. Introduction 7
2.1.2. Subjects and Methods 8
2.1.3. Results 12
2.1.4. Discussion 14
2.2 Gastric Volumes in Diabetes Mellitus and Health: Vagal Function
and Effects of Glucagon-Like Peptide-1
2.2.1 Introduction 17
2.2.2 Patients and Methods 18
2.2.3 Results 20
2.2.4 Discussion 22
3. Figures 26
4. Tables 35
5. Final Remarks 44
6. Summary and Future Studies 49
7. Acknowledgments 50
8. References 51
Delgado-Aros S. - 3 -
1. General Introduction Glucagon-like peptide-1
Glucagon-like peptide-1 (GLP-1) is a peptide derived from a specific proteolytic
processing of the proglucagon molecule in L cells of the jejunum and colon 1, 2. Different
forms of this peptide are generated from this proteolysis: GLP-1-(1-37), which is
biologically inactive, and GLP-1-(7-37) or GLP-1-(7-36)amide, which are the
biologically active forms 3-5. GLP-1 will be used in this text to refer to the short, active
forms of the molecule.
This peptide is being proposed as a new treatment in people with type II diabetes
since it stimulates insulin gene expression 3 and potentiates glucose-induced insulin
release from pancreatic β cells 6. It has been shown that exogenous administration of
GLP-1 helps to control postprandial hyperglycemia in type II diabetes 7.
Besides its role regulating endocrine secretion from the pancreas, GLP-1 has been
shown to regulate several other gastrointestinal functions, for example it decreases gastric
acid secretion 8, gastric emptying rate 9, 10 and intestinal transit 11.
GLP-1 may also play a role in the regulation of food intake. There is evidence in
animal models and humans that administration of exogenous GLP-1 decreases food
consumption over the short term and increases postprandial satiety 12-16. Abdominal
symptoms, such as nausea, have also been described after high doses of exogenous GLP-
1 infusion 17, 18.
It is still not clear how these effects of GLP-1 are mediated. In vitro studies have
demonstrated that direct binding of the peptide to the GLP-1 receptor in the pancreatic β
cells is followed by insulin secretion 19. Nevertheless, GLP-1 is rapidly inactivated when
it is released into the circulation 20, being the in vivo half life of the active GLP-1 limited
to approximately 1 minute. The rapid inactivation of GLP-1 in the circulation makes it
unlikely that active GLP-1, released from the intestinal tract, can reach distant organs
such as the pancreas. It is more likely that effects of GLP-1 on distant organs are neurally
mediated. In vitro, binding of GLP-1 to receptors in gastric parietal cells stimulates acid
production 21. In contrast, in vivo administration of GLP-1 produces inhibition of gastric
acid secretion 8. The effects of GLP-1 on gastric acid and pancreatic secretions are
abolished after vagotomy in animals and humans 22, 23. These observations suggest that
Delgado-Aros S. - 4 -
the effects of GLP-1 are most likely to be vagally mediated. It has also been shown that
GLP-1 modulates the vagally-mediated delay in gastric emptying in response to
intraluminal nutrients 24. Overall, these data suggest an inhibitory effect of GLP-1 on
vagally-mediated reflexes in the upper gastrointestinal tract.
Gastric Accommodation and Vagal Function
The vagus nerve is known to modulate most of gastrointestinal functions, including
secretion, absorption or motor functions. The abdominal vagus is known to carry mostly
afferent (sensory) fibers whereas only 10% are efferent motor fibers 25. Cell bodies of
visceral vagal fibers lie in vagal ganglia and their central processes terminate
predominantly in the nucleus of the solitary tract (NST). From the NST afferent fibers
connect to several central regions (such as the hypothalamus, amygdala and limbic
cortex). They also convey signals to the dorsal motor nucleus of the vagus (DMNV) from
which efferent fibers convey messages to the NST and to the gut wall 26. These
anatomical connections provide the basis for the vago-vagal reflexes that are crucial for
the adequate control of upper gastrointestinal functions. (See Introductory Schema)
The accommodation or relaxation of the stomach in response to meal ingestion has
been shown to involve the vagus nerve since vagal denervation impairs gastric relaxation
in animals 27-29 and humans 30. This vagally-mediated reflex prevents the increase in
intragastric pressure when food and fluid enter in the stomach 31, avoiding the
development of postprandial symptoms such as nausea, bloating, pain or vomiting in
health. There is also evidence suggesting that this gastric postprandial reflex plays an
important role in the regulation of satiation 32, 33, one of the final signals that arise during
meal ingestion and that contribute to meal termination 34.
Study Hypotheses
Our first hypothesis was that GLP-1 induces inhibition of gastric accommodation
reflex, as part of its general inhibition of vagally-mediated reflexes in the gastrointestinal
tract, and that this effect could partly explain the effects of GLP-1 on food consumption
and postprandial symptoms and satiety. To test this hypothesis, we compared, in healthy
volunteers, the effects of intravenous infusion of GLP-1 and placebo, on gastric
postprandial accommodation, postprandial response of plasma human pancreatic
polypeptide (a surrogate marker of vagal abdominal function), the volume of a nutrient
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liquid meal (Ensure) ingested at maximum satiation, postprandial symptoms and gastric
emptying of Ensure. Measurements of gastric emptying were included to evaluate the
potential confounder of delayed gastric emptying when assessing the effects of gastric
accommodation on satiation and postprandial symptoms.
The results of the first study showed that GLP-1 does not inhibit postprandial gastric
accommodation; on the contrary, it increased fasting and postprandial gastric volumes.
This was accompanied by a marked inhibition of the normal postprandial increase of the
pancreatic polypeptide, a hormone whose release is under vagal cholinergic control 35.
Since gastric tone (contraction) is maintained by vagal cholinergic (excitatory) input 27, 36,
the results observed in our first study suggested that GLP-1 could induce gastric
relaxation by inhibition of vagal (excitatory) cholinergic pathways during fasting and
postprandially. (See Introductory Schema)
If this hypothesis was true, one would predict that in the presence of vagal
dysfunction or vagotomy, GLP-1 would not induce gastric relaxation. To test this second
hypothesis we studied the effect of the same intravenous infusion of GLP-1, compared to
placebo, on fasting and postprandial gastric volumes in a sample of subjects with diabetes
affected with vagal neuropathy.
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DMNV
NST
V nervespinaltract
VagusNerve
AfferentsEfferents- Nitrergic : Inhibit gastric tone- relaxationEfferents- Cholinergic: Activate gastric tone- contraction
Introductory Schema
NucleusAmbiguus
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2.1 Effect of GLP-1 on Gastric Volume, Emptying, Maximum Volume
Ingested, and Postprandial Symptoms in Humans 2.1.1. Introduction
Glucagon-like peptide-1 (7-36) amide (GLP-1) is produced by the processing of
proglucagon in enteroendocrine L cells of the intestinal mucosa. It is released in response
to meal ingestion 37, 38, exerting a glucose-dependent effect on β cells of the pancreas
enhancing insulin release. GLP-1 also has an inhibitory effect on the pancreatic α cells,
reducing glucagon release 39, 40. These properties provide the rationale for reducing
glycemia and for its use in diabetes mellitus 7, 10, 41.
However, GLP-1 also exerts several effects on the upper digestive tract: inhibition
of gastric acid and pancreatic exocrine secretions 23, 42, 43, delay in gastric emptying for
liquids and solids in health 9 and diabetes 10. The latter may result from enhanced pyloric
tone or diminished antroduodenal motility during the interdigestive and fed states in
health 44. Preliminary data also indicate relaxation of the proximal stomach in response to
intravenousGLP-1 during fasting 45. GLP-1 has been reported to also reduce the amount
of food and fluid consumed, to reduce hunger and enhance the feeling of fullness in
health 16 and diabetes 14 but its effects on postprandial symptoms are unclear.
Previous studies showed dose-related, reversible inhibition of human pancreatic
polypeptide (HPP) release in response to a meal after subcutaneous or intravenous
infusion of GLP-1. GLP-1 also inhibits centrally-induced pancreatic and gastric acid
secretions 42, and these effects are lost after abdominal vagotomy in humans 22 and pigs 23, suggesting an inhibition of efferent vagal-cholinergic function.
Postprandial gastric accommodation is a vagally-mediated reflex 46, 47. Impaired
gastric accommodation is an important cause of postprandial symptoms 48-51. We
hypothesized that GLP-1 diminishes the postprandial gastric accommodation response by
inhibition of vagal function, reducing maximum volume ingested and increasing
postprandial symptoms. The aims of this study were to compare the effects of GLP-1 on
postprandial gastric volumes, gastric emptying, maximum tolerated volume of a nutrient
liquid meal, postprandial symptoms and vagal function in healthy volunteers.
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2.1.2. Subjects and Methods
Study Population
Healthy volunteers over 18 years of age were recruited from the local community
by public advertisement. Exclusion criteria included: pregnant or breastfeeding females,
prior abdominal surgery other than appendectomy or tubal ligation; positive symptoms on
an abridged bowel disease questionnaire; present or previous chronic gastrointestinal
illness; systemic disease or use of medications that may alter gastrointestinal motility.
Study Design
This study was approved by the Mayo Institutional Review Board. Eligible
volunteers gave their written informed consent and were randomized to receive either
GLP-1 (Bachem, San Diego, CA) as an infusion of 1.2 pmol/kg/min over 60 minutes, or
saline infusion (placebo) in a double blind design.
The study was performed on two consecutive days. On the first day (Protocol 1),
subjects underwent assessment of gastric volumes and measurements of fasting and
postprandial glucose and HPP. On the second day (Protocol 2), maximum tolerated
volume (MTV), scintigraphic gastric emptying and postprandial symptoms were
assessed. GLP-1 or saline (placebo) was infused for sixty minutes on both occasions.
GLP-1
GLP-1 was infused at a rate of 1.2 pmol/Kg/min. Previous studies have
demonstrated that steady state levels are achieved within ~30 minutes from the onset of
theinfusion 41. Therefore, the physiological measurements of gastric accommodation,
emptying and satiety, as well as the plasma levels of glucose and HPP, were done under
steady levels for a total of at least 30 minutes.
We used an infusion rate of GLP-1 of 1.2 pmol/Kg/min since it has been
previouslyshown to affect gastrointestinal function and satiety in humans 41, 43-45. Higher
rates of GLP-1 infusion may cause gastrointestinal distress 17. 99mTc-SPECT method to measure gastric volume
We used a noninvasive method to measure fasting and postprandial gastric
volumes in humans using single photon emission computed tomography (SPECT) 52.
This method has been recently validated in vitro and in vivo 53. In healthy volunteers,
simultaneous measurements of postprandial gastric volume changes with SPECT and the
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barostat balloon device were strongly correlated (r = 0.9) 53.The latter is currently
considered the gold standard for the measurement of gastric accommodation. We have
also showed the high intraobserver reproducibility of this technique to measure gastric
volumes 54. In dyspeptic patients, the SPECT technique has reproduced the changes in
gastric volume 55 obtained by using the barostat device 33.
The method has been described in detail elsewhere 52, 55. Briefly, 10 minutes after
intravenous injection of 99mTc-sodium pertechnetate, dynamic tomographic acquisition of
the gastric wall was performed using a dual-head gamma camera (Helix SPECT System,
Elscint, Haifa, Israel) which performs orbits of 360° around the supine participant and
takes 10 minutes per orbit. Each orbit leads to a three-dimensional rendering of the
stomach using the AVW 3.0 (Biomedical Imaging Resource, Mayo Foundation,
Rochester, MN) image processing libraries. This was accomplished by identifying the
stomach in the transaxial SPECT images and separating it from the background noise
using a semi-automated segmentation algorithm (Fig. 1).
A customized algorithm was developed to estimate the volume of the proximal
stomach; this algorithm estimates the longest axis of the reconstructed stomach and
divides it into a proximal two-thirds and distal one-third. The volume corresponding to
the length of the proximal two-thirds was then obtained.
Protocol 1: Measurement of gastric volumes, plasma levels of glucose and HPP
After an 8 hour period of fasting, patients lay down on the SPECT camera and the
10mCi 99mTc-sodium pertechnetate was injected intravenously. Ten minutes later, a first
orbit (360º over 10 minutes) was performed for baseline (pre-infusion) tomographic
images (Fig. 2).
Infusion of 1.2 pmol/Kg/min GLP-1 or saline placebo was started. After 30
minutes' infusion (time to achieve steady levels), a second image was obtained over 10
minutes to assess effects of the GLP-1 on fasting gastric volumes.
A nutrient liquid meal (Ensure®: 300 mL, 316 Kcal, 7.6g fat, 50.6g carbohydrate
and 11.4g protein) was ingested over 3 minutes, and two further 10 minute images were
obtained to measure postprandial gastric volumes.
Blood samples were taken at baseline (pre-infusion), after 30 and 40 minutes’
infusion (fasting period), and 10 and 20 minutes postprandially for measurement of
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glucose and HPP. Plasma glucose concentrations were measured using a glucose oxidase
method (using a glucose analyzer). Plasma levels of HPP were analyzed using a
radioimmunoassay kit 56, 57.
Protocol 2: Measurement of MTV, gastric emptying and postprandial symptoms
To compare effects of GLP-1 and placebo on MTV (that is, the volume ingested
until maximum satiety is reached), we adapted the method used by Tack et al. 33,
appending a scintigraphic evaluation of gastric emptying by radiolabeling Ensure®
ingested during the test (Fig. 3).
After 30 minutes of GLP-1 or saline placebo infusion to achieve steady state,
subjects were asked to ingest Ensure® at a constant rate (30 mL per minute) by refilling a
glass with a perfusion pump, and drinking at the filling rate. Participants scored their
level of satiety during the drink test using a graphic rating scale graded 0-5 (0 = no
symptoms; 5 = maximum or unbearable satiety). Participants were told to stop meal
intake when a score of 5 was reached. The total volume ingested was the MTV.
To assess gastric emptying, the second glass of Ensure® was radiolabeled with 50
µCi of 111In-DTPA and 1-minute duration scans of the abdomen were obtained at 10-
minute intervals for the first 30 minutes and then at 15-minute intervals until at least 50%
of the meal was emptied, or for a maximum of 3 hours after the meal.
Thirty minutes after completing ingestion of the Ensure®, participants were
requested to score their postprandial symptoms (nausea, bloating, fullness, pain) using a
10cm visual analog scale anchored with the words unnoticeable and unbearable at the left
and right ends of the lines. This symptom assessment is consistent with previous studies
in the literature 58.
Data Analysis
Gastric volumes
Total and proximal gastric volume at baseline (pre-infusion), fasting and during
two postprandial periods (0-10 minutes and 10-20 minutes) were measured; the
postprandial gastric volume was calculated from the average of the two postprandial
volumes. Volume change from baseline (pre-infusion) to fasting and postprandial periods
were assessed as differences and as ratios over baseline volumes (Fasting Difference=
Fasting Volume-Baseline volume; Fasting Ratio= Fasting Volume/Baseline Volume;
Delgado-Aros S. - 11 -
Postprandial Difference= Postprandial Volume-Baseline Volume; Postprandial Ratio=
Postprandial Volume/Baseline Volume).
MTV and postprandial symptoms
The total volume ingested (MTV) was recorded. The aggregate postprandial
symptoms score (thirty minutes after completing ingestion of Ensure®) was calculated as
the sum of VAS scores for each postprandial symptom (maximum 400).
Gastric emptying
Gastric emptying during the drink test was measured by scintigraphy by
radiolabeling the second glass of Ensure for all participants and as described above. The
primary endpoint for assessment of effects on gastric emptying was the proportion
emptied at 30 minutes, which corresponded with the time when the GLP-1 or placebo
infusion was completed. This time was selected in view of the very short half-life of
infused GLP-1 estimated as ~5 minutes 59. At this time point all the participants had
ingested approximately the same volume since the rate of ingestion of the nutrient liquid
meal was standardized and all the participants, except one, were still drinking at 30
minutes. Four of the participants who reached full satiety at that point did not completely
drink the last glass of Ensure® (200mL) and had slightly less volume and caloric intake:
3 were in the GLP-1 group (875, 822 and 772 mL), and 1 in the placebo group (882 mL).
Thus, the volume and caloric intake was identical for nineteen of the participants. The
secondary endpoint was the proportion emptied at 90 minutes (one hour after the infusion
ended); this was intended to determine whether there were longer lasting effects of the
infused hormone.
Plasma glucose and HPP
Fasting and postprandial plasma levels of glucose and HPP were calculated from
the average of the two fasting measurements and the two postprandial measurements
respectively. Changes in the levels of glucose and HPP from baseline to fasting and to
postprandial periods were assessed by subtracting baseline (pre-infusion) values from
fasting and postprandial levels.
Statistical Analysis
Unpaired t-test was used to compare absolute gastric volumes as well as the
volume differences and ratios between GLP-1 and placebo groups. Wilcoxon rank sum
Delgado-Aros S. - 12 -
test was used to compare the variables that were not normally distributed: maximum
tolerable volume (MTV), the aggregate postprandial symptoms score, the gastric
emptying at 30 and 90 minutes, and change in plasma levels of glucose and HPP. Prior to
the study, the estimated sample size for 80% power to detect a 25% difference in the
primary endpoint (postprandial gastric volume) in response to GLP-1 compared to
placebo was 12 per group (α = 0.05). All the tests were two-tailed and results are
presented as medians and interquartile ranges (IQR).
2.1.3. Results
Study Conduct and Participants
Twenty-four healthy volunteers were studied (13 in the GLP-1 group and 11 in
the placebo group). We were not able to obtain peripheral blood samples from two
participants; accurate assessment of gastric emptying was not possible for technical
reasons in one participant. Missing data excluded these individuals from specific
comparisons; however, data for all 24 participants were used when available that is, in all
comparisons except where indicated above. There were no statistically significant
differences among demographic and baseline variables between the two study groups
(Table 1).
Total Gastric Volumes
Figure 4 shows examples of the stomach reconstructions at baseline (pre-
infusion), fasting and postprandially in the GLP-1 and placebo groups. Table 2 shows the
data for total gastric volumes, differences in volumes and ratios. The fasting volume was
significantly greater in the group that received GLP-1 (312 mL, IQR: 253 to 365)
compared to the placebo group (225 mL, IQR: 185 to 239; p=0.002).
The difference between fasting and baseline volume was 80 mL (IQR: 61 to 128)
for the participants who received GLP-1 and 17 mL (IQR: -21 to 25) for those who
received placebo (p=0.005). The ratio of fasting over baseline volume was also greater
for the GLP-1 group (1.48 IQR: 1.26 to 1.60) than for the placebo group (1.08 IQR: 0.90
to 1.12; p=0.003).
Postprandial volumes were also greater in the GLP-1 group (848 mL, IQR: 789 to
899) compared to placebo group (651mL, IQR: 602 to 801; p=0.004). The difference
Delgado-Aros S. - 13 -
between postprandial and baseline volume was 608 mL (IQR: 532 to 671) for GLP-1
group, and 435 mL (IQR: 401 to 549) for placebo group (p=0.008). No significant
differences were found between groups when comparing total gastric volume ratios
postprandially, 3.53 (IQR: 3.19 to 4.39) for the GLP-1 group and 3.14 (IQR: 2.79 to 3.61)
for the placebo group (p= 0.15).
Proximal Gastric Volumes
Table 3 shows gastric volumes, differences and ratios for the proximal stomach in
the two groups. No significant differences were found when comparing fasting absolute
volumes, differences between fasting and baseline volumes or the fasting/baseline ratios.
In contrast, the postprandial proximal volumes were significantly greater in the
GLP-1 group (629 mL, IQR: 581 to 647) than the placebo group (455 mL, IQR: 338 to
618) (p<0.0001). The absolute difference between postprandial and baseline volume was
also significantly greater in the GLP-1 group (461 mL, IQR: 400 to 553) compared to the
placebo group (302 mL, IQR: 223 to 389; p= 0.0003). There was a trend towards a
greater postprandial ratio in the GLP-1 group (5.77, IQR: 3.41 to 7.45) compared to the
placebo group (3.72, IQR: 2.84 to 4.81; p=0.08).
MTV and Postprandial Symptoms
As shown in Table 4, the median volume ingested to reach full satiety was 1119
mL (IQR: 874 to 1546) for GLP-1 group and 1350 mL (IQR: 1082 to 1606) for placebo
group (p=0.16). The individual values are shown in figure 5.
The aggregate postprandial symptom score was 185 (IQR: 121 to 250) in the
GLP-1 group and 169 (IQR: 121 to 199) in the placebo group (p=0.54). No differences
were found when comparing each of the symptoms separately (nausea, bloating, fullness
and abdominal pain; see Table 4).
Gastric Emptying of Nutrient Liquid
One volunteer, subsequently shown to be in the GLP-1 group, vomited after the
satiety test was completed. These data were excluded from the analysis of gastric
emptying.
Figure 6 shows the gastric emptying of the radiolabeled liquid nutrient meal. The
proportion emptied was significantly lower for the GLP-1 group at the point the infusion
ended, at 30minutes (7%, IQR: 3.5 to 19 vs. 23% IQR: 14 to 23, respectively; p=0.008).
Delgado-Aros S. - 14 -
However, this effect was transient; one hour after the infusion ended, the proportion
emptied was not different for the two groups, 21% (IQR: 14.5 to 38) for the GLP-1 group
vs. 35% (IQR: 21.75 to 38.25) for the placebo group (p=0.28).
Plasma Levels of Glucose and HPP
During fasting, the glucose change relative to baseline (pre-infusion) was -12.3
mg/dL (IQR: -19.1 to -8) for GLP-1 group and 0.5 mg/dL (IQR: -2.3 to 3.3) for placebo
group (p=0.0002). The postprandial increase in glucose levels was -9.3 mg/dL (IQR: -
18.5 to -6.4) for the GLP-1 group and 19.3 mg/dL (IQR: 15.9 to 26.6) for the placebo
group (p<0.0001; Fig. 7).
The fasting HPP change relative to baseline was similar in the two groups: -12.0
pg/mL (IQR: -22.5 to -2.0) for GLP-1 and -0.25 pg/mL (IQR: -14.4 to 31.4) for placebo
(p= 0.12). However, GLP-1 reduced significantly the postprandial increase in HPP
levels: 6.5 pg/mL (IQR: -22.4 to 6.9) for GLP-1 compared to 119.8 pg/mL (IQR: 60.1 to
357.0) for placebo (p=0.0001).
2.1.4. Discussion
The results of the present study suggest that GLP-1 increases gastric volume
during fasting and in the postprandial period and retards gastric emptying. These effects
are not associated with changes in maximum volume of Ensure® tolerated or in
postprandial symptoms.
In this study, we confirmed the delay of gastric emptying for liquid meals during
intravenous infusion of GLP-1 in healthy subjects. Schirra et al. 9 had previously
reported that an isolated subcutaneous injection of either 125 or 250 pmol/Kg of GLP-1
delays the emptying of a 300Kcal mixed liquid meal. The retarding effect of GLP-1 on
gastric emptying is transient, and the post-infusion emptying of the liquid meal (as
assessed by the proportion emptied at 90 minutes, that is, one hour after the infusion
ended) was not different in the two groups. This observation is consistent with the short
biological activity of the hormone (~5 min). The mechanism by which GLP-1 delays
gastric emptying of liquids is unclear. The reported inhibition of antroduodenal motility
during the postprandial state 9, 44 and the increase in isolated pyloric pressure waves
(IPPWs) may contribute to delayed emptying of solids. However, gastric emptying of
Delgado-Aros S. - 15 -
liquids is thought to depend on fundic pressure 60 and to be less influenced by antral
motility 61, 62. An alternative mechanism for delayed gastric emptying of liquids is that
the GLP-1-induced increase in postprandial gastric volume was associated with a
decrease in fundic tone. Previous studies on GLP-1 have shown decreased fasting fundus
tone 45.
GLP-1 decreases the feeling of hunger before meals and reduces food and fluid
intake in healthy subjects 13, 16 and in diabetic 14 and nondiabetic 15 obese patients.
However, no effects of GLP-1 on postprandial symptoms have been reported. In this
study, we observed no differences in the maximum tolerated volume and aggregate
postprandial symptoms scores or in individual symptoms of nausea, bloating, fullness or
pain. It might be expected that increased gastric volume could allow the ingestion of a
larger volume before reaching satiation and, possibly reduce the likelihood of developing
postprandial symptoms. Failure to observe this could be explained by the marked
inhibition of gastric emptying by GLP-1. Another possible explanation is that GLP-1
might regulate food intake independently of its motor effects. Data from animal studies
suggest a central site of action of the effect of GLP-1 on reduced food consumption,
unrelated to a change in gastric functions 12, 24. Thus, we postulate that GLP-1 sensory
effects might also be centrally mediated in humans.
Wank et al. 45 showed that slightly lower infusion rates of 0.3 and 0.9
pmol/Kg/min of GLP-1 diminished fasting gastric tone recorded with an electronic
barostat device. We confirmed this finding in our study using SPECT and expanded the
knowledge base by showing the effect is observed in both proximal and whole stomach.
Prior to our study, the effects of GLP-1 on postprandial gastric volumes or
accommodation had not been reported. In our study, greater postprandial gastric volumes
(proximal and whole stomach) with GLP-1 were demonstrated compared to placebo,
using a validated method that images the gastric wall, not the intragastric content. Hence,
this method is independent of the volume and the rate of emptying of the meal. Our
method does not measure tone and therefore cannot measure relaxation of the stomach.
However, since the intragastric pressure is subject to the positive intra-abdominal
pressure and to equilibration with atmospheric pressure via the belching reflex, and since
these conditions were not altered before and after the meal, the postprandial increase in
Delgado-Aros S. - 16 -
volume, measured by SPECT, constitutes a measure of the gastric accommodation, which
is enhanced by intravenous infusion of GLP-1.
The mechanisms by which GLP-1 increases gastric volume are unclear. It is
known that, during fasting, gastric tone is maintained via vagal cholinergic input and that
�2-adrenergic and nitrergic pathways induce gastric relaxation 47, 63. During the fed
state, gastrointestinal motility is partly controlled by nonadrenergic, noncholinergic
(NANC) vagal pathways 64, and nitric oxide (NO) modulates the postprandial
accommodation response 46, 65. Our study starts to explore the mechanism for the
enhanced postprandial gastric volume in response to GLP-1. Thus, we have shown that
the effect of GLP-1 on postprandial gastric volume is accompanied by a marked
inhibition of the normal postprandial increase of human pancreatic polypeptide. The
latter is a hormone of the endocrine pancreas that is under cholinergic control 35. The
effect of GLP-1 on the human postprandial pancreatic polypeptide response has been
previously shown to be independent of gastric emptying 10, 44. This suggests that the
delay in gastric emptying by GLP-1 is not the cause of the inhibition of pancreatic
polypeptide release. Therefore, our data are consistent with GLP-1 inhibition of efferent
vagal-cholinergic function.
The increased gastric volume observed with GLP-1 may result from inhibition of
cholinergic innervation during fasting and postprandially. An alternative hypothesis is
that GLP-1 enhances gastric volumes by activation of vagal nitrergic pathways, which
mediate the normal postprandial accommodation response.
In conclusion, we have shown that GLP-1, a novel agent in the treatment of
diabetes and obesity, increases the fasting and postprandial volume of the stomach,
transiently retarding gastric emptying without increasing postprandial symptoms in
healthy subjects. The current study suggests that GLP-1 induces gastric relaxation
through a mechanism that involves the vagus nerve. To test this hypothesis we performed
the second study presented in this thesis.
Delgado-Aros S. - 17 -
2.2 Effects of Glucagon-Like Peptide-1 and Feeding on Gastric Volumes
in Diabetes Mellitus with Cardio-Vagal Dysfunction 2.2. 1 Introduction
Glucagon-like peptide-1 (GLP-1), a peptide released after meal intake 39, has
several effects on gastric and pancreatic functions by a mechanism that appears to
involve the vagus nerve. The presence of nutrients in the small intestine results in
vagally-mediated inhibition of gastric emptying. This response is abolished by the GLP-1
receptor antagonist exendin-(9,39) 28. Exogenously administered GLP-1 inhibits gastric
acid secretion and gastric emptying 43. These effects are respectively abolished by vagal
afferent denervation in rats 24 and in vagotomized patients 22. In the first study presented
in this thesis, we showed that GLP-1 increases gastric volume during fasting and
postprandially in healthy subjects. This response was associated with inhibition of the
pancreatic polypeptide response over the first 20 minutes after the meal. Pancreatic
polypeptide response to feeding is dependent on vagal activation since vagal blockade
completely abolishes the response 66-70. However, the effect of GLP-1 on gastric volume
in the presence of vagal dysfunction is unknown.
Cholinergic and nitrergic vagal pathways are involved in the regulation of gastric tone in
animals 27-29, 60, 71 and humans 30, 36, 72, 73. Vagal participation in the gastric relaxation
response to a meal is mediated through nonadrenergic noncholinergic fibers 74-76.
However, there is increasing evidence suggesting that the control of gastric tone and the
gastric relaxation after a meal may recover with passage of time after vagal denervation 30, 77, 78. In elegant animal studies by Takahashi and Owyang, it was shown that this
recovery of gastric accommodation is neurally-mediated and appears not to involve
sympathetic pathways 28 suggesting adaptive control of gastric tone by the enteric
nervous system (ENS).
However, in a human model of vagal neuropathy such as diabetic vagal
neuropathy, it is not yet known whether the gastric volume response to a meal is
preserved, since previous data are controversial with some studies showing diminished
mean gastric accommodation in diabetic patients with vagal neuropathy, while individual
accommodation responses were within the normal range in some of the patients evaluated 50, 79, 80.
Delgado-Aros S. - 18 -
In this study, we hypothesized that gastric volume response to GLP-1 is abolished
in diabetic patients with vagal neuropathy. To test this hypothesis, we studied the
responses of fasting and postprandial gastric volume to placebo and GLP-1 in diabetic
patients with evidence of cardio-vagal neuropathy. We also compared gastric volumes in
diabetic patients on placebo to those in the healthy subjects who participated in the first
study presented in this thesis.
2.2. 2 Patients and Methods
Study Population
Healthy subjects
Volunteers over 18 years of age recruited from the local community by public
advertisement. Exclusion criteria included: pregnant or breast-feeding females, prior
abdominal surgery other than appendectomy, laparoscopic cholecystectomy or tubal
ligation; positive symptoms on an abridged bowel disease questionnaire; present or
previous chronic gastrointestinal illness; systemic disease or use of medications that may
alter gastrointestinal motility.
Diabetic subjects
Diabetic patients (over 18 years of age) diagnosed at Mayo Clinic, Rochester
(MN) with a positive diagnosis of cardiovagal neuropathy were recruited from the local
community by public advertisement. Exclusion criteria were the same as for healthy
volunteers, except for the presence of diabetes.
Cardiovagal neuropathy serves as a surrogate of abdominal vagal neuropathy;
pathological and functional observations show that changes in the vagus nerve start
distally and progress cranially 81-83. Thus, when the branches of the vagus that innervate
the heart are affected, the more distal branches of the vagus, such as the gastric nerve, are
affected 84-88.
Cardiovagal neuropathy was based on heart rate (HR) variability in response to
deep breathing. Heart rate was recorded using a standard ECG monitor. The subject
performed deep breathing at a respiratory rate of 6 per minute for six cycles. HR
variability was analyzed as the average HR change for the six consecutive cycles. This is
a widely used test to noninvasively assess the cardiovagal function in human subjects 89-
91.
Delgado-Aros S. - 19 -
Study Design
The studies were approved by the Mayo Institutional Review Board, and all
eligible volunteers gave written informed consent before enrollment in the study.
The study performed in healthy volunteers followed a randomized, placebo-
controlled, double-blind, parallel-group design as described before. Eligible volunteers
were randomized to receive either GLP-1 or saline infusion (placebo) for 60 minutes.
Participants with diabetes were randomized in a placebo-controlled, double-blind,
crossover design study and received GLP-1 or saline infusion (placebo) for 60 minutes on
two separate days (at least 48 hours apart). Diabetic subjects underwent assessment of
gastric volumes under euglycemic conditions.
GLP-1
GLP-1 (Bachem, San Diego, CA) was infused during 60 minutes at a rate of 1.2
pmol/Kg/min, a dose previously shown to affect gastrointestinal function without causing
side effects in healthy volunteers 43, 44. Gastric volume measurements were started after
30 minutes of infusion when GLP-1 steady levels in plasma are achieved 41.
Measurement of gastric volumes using 99Tc-SPECT (Fig. 8)
Participants were studied after at least 6 hours of fasting. The diabetic patients
were specifically asked to have their last meal in a liquid form. Blood glucose was
monitored by means of a reflectance meter prior to commencing and throughout the
study. Intravenous insulin was used as necessary to maintain glucose levels in the normal
range (60-120 mg/dL). Patients lay down on the SPECT camera, and the 10mCi 99mTc-
sodium pertechnetate was injected intravenously. Ten minutes later, a first orbit (360º over 10 minutes) was performed for baseline (pre-infusion) tomographic images.
Infusion of 1.2 pmol/Kg/min GLP-1 or saline placebo was started. After 30
minutes' infusion, a second image was obtained over 10 minutes to measure the effects of
the GLP-1 compared to placebo on fasting gastric volumes.
A nutrient liquid meal (Ensure®: 300 mL, 316 Kcal, 7.6g fat, 50.6g carbohydrate
and 11.4g protein) was ingested over 3 minutes, and one further 10 minute image was
obtained to measure postprandial gastric volume.
Data Analysis
Gastric volumes
Delgado-Aros S. - 20 -
Total and proximal gastric volumes at baseline (pre-infusion), fasting and during
10 minutes postprandially were measured. Volume changes from baseline (pre-infusion)
to fasting and postprandial periods were assessed as differences and as ratios over
baseline volumes (Fasting Difference= Fasting Volume-Baseline volume; Fasting Ratio=
Fasting Volume/Baseline Volume; Postprandial Difference= Postprandial Volume-
Baseline Volume; Postprandial Ratio= Postprandial Volume/Baseline Volume).
Statistical Analysis
Mann-Whitney and Wilcoxon signed rank sum tests were used for inter- and
intra-individual comparisons respectively. Based on the measurements of the increase in
gastric volumes in healthy subjects in response to GLP-1, a sample size of 7 provided
94% power to detect the same increase in diabetic patients using a crossover design and
one-side alpha of 0.05. We used a one-tailed test in this case since the null hypothesis
was unidirectional, that is, that GLP-1 does not increase the gastric volume in diabetic
patients. All other comparisons were based on two-tailed tests, and the significance level
was set at 0.05. The results are presented as medians and interquartile ranges (IQR).
2.2. 3 Results
Study Conduct and Participants
Seven diabetic patients with a diagnosis of cardiovagal neuropathy were studied.
In the healthy studies 24 healthy volunteers participated (13 in the GLP-1 group and 11 in
the placebo group). Demographic variables for both study populations are presented in
Table 5. Of the 7 diabetic patients, 5 had insulin-dependent diabetes mellitus (IDDM)
and 2 non-insulin-dependent diabetes mellitus (NIDDM); 5 had diabetic retinopathy; 5
had peripheral somatic neuropathy; 4 had erectile dysfunction; and 3 renal impairment.
One of the diabetic patients had chronic diarrhea, which was attributed to autonomic
dysfunction after ruling out other possible diagnoses. No other gastrointestinal symptoms
were reported by any of the patients.
Two diabetic patients received insulin before starting the placebo study and two
other patients received insulin before starting the GLP-1 study to correct fasting
hyperglycemia (plasma glucose >180 mg/dL). Insulin was stopped during the study in all
the patients. One diabetic patient received intravenous glucose during the study to
Delgado-Aros S. - 21 -
correct insulin-induced hypoglycemia (plasma glucose <80).
Effect of GLP-1 on Gastric Volumes in Diabetes with Vagal Neuropathy
Total gastric volumes
Table 6 shows the effects of placebo or GLP-1 infusion on total gastric volumes at
baseline, during fasting, and ten minutes postprandially. The differences in volumes and
ratios post-drug over baseline volumes are also presented. GLP-1 did not increase fasting
or postprandial gastric volumes in diabetic patients. Data demonstrating significant
effects of GLP-1 in healthy volunteers from our previous study are included in the table
for comparison.
Proximal gastric volumes
Table 7 shows the effects of placebo or GLP-1 infusion on proximal gastric
volumes at baseline, during fasting, and ten minutes postprandially. The differences in
volumes and ratios post-drug over baseline volumes are also presented. As observed
when assessing total volumes, GLP-1 did not increase the fasting or postprandial volume
of the proximal stomach.
Figure 9 shows gastric volume changes fasting and postprandially in diabetic
patients during placebo or GLP-1 infusion and includes results in healthy individuals
from our previous study for comparison.
Gastric Volumes During Fasting and After a Standard Meal in Health and
Diabetes with Vagal Neuropathy
Observations during placebo treatment studies were used to compare gastric
volume during fasting and the gastric volume response to a standard meal in diabetic
patients with vagal neuropathy with those observed in healthy controls. The total and
proximal gastric volumes measured during fasting and postprandially in diabetics with
vagal neuropathy under placebo treatment (n=7) were not significantly different
compared to those in healthy subjects under placebo treatment (n=11). (Table 8)
Figure 10 shows examples of the stomach reconstructions during fasting and
postprandially in a healthy subject and a diabetic patient.
Plasma Glucose Levels
Plasma glucose levels in diabetic patients were similar on both study days
(Fig. 11) and within the normal range during fasting (100 mg/dL IQR:89; 124) and
Delgado-Aros S. - 22 -
postprandially (111 mg/dL IQR: 90; 127).
2.2. 4 Discussion
The diabetic patients evaluated in our study had fasting gastric volumes that were
not significantly different from those of healthy controls. Our study also shows that
exogenous GLP-1 increases gastric volume in healthy individuals, but not in diabetic
patients with vagal neuropathy who retain the ability to increase the gastric volume in
response to a meal. These data strongly suggest that the effect of GLP-1 on gastric
volume is dependent on vagal function. This interpretation is consistent with previous
studies that suggested that GLP-1 inhibition of gastric acid secretion and emptying is
mediated through the vagus 22, 24 and with our data from the first study presented in this
thesis, showing that GLP-1-induced increase in gastric volume is associated with vagal
inhibition.
Gastric relaxation in response to a meal results in an increase of gastric volume to
accommodate ingested food, without an increase in intragastric pressure. This prevents
the development of symptoms caused by the increase in intragastric hydrostatic pressure
from the meal and gastric secretions. In animal and in vitro studies, this reflex response
has been thoroughly characterized. When the food arrives in the stomach, vagal afferent
terminals in the gastric wall convey the signal to the nucleus of the solitary tract (NST) in
the dorsal vagal complex (DVC). These first order afferents synapse with neurons in the
dorsal motor nucleus of the vagus (DMNV) in the DVC 92. From the DMNV,
nonadrenergic noncholinergic (NANC) vagal efferents 93 reach the stomach inducing
gastric relaxation. Afferents from the NST in the DVC connect to several central regions
(such as hypothalamus, amygdala and limbic cortex) 94 that project back to the DVC 95.
The DVC also binds hormones and neuropeptides such as GLP-1 96. All these neural and
hormonal influences are important to modulate gastrointestinal function and eating
behavior 88, 97.
In the denervated gut, it is well known that the enteric nervous system (ENS) can
elicit the motility patterns that characterize the fasting and postprandial states 98-100, as
well as intrinsic reflexes such as the peristaltic reflex stimulated by a food bolus 101, 102.
King and Szurszewski have demonstrated that a number of entero-enteric reflexes evoked
Delgado-Aros S. - 23 -
by distension relay through the prevertebral ganglia 103. In vitro, gastric relaxation in
response to distension is maintained in the denervated and vascularly-isolated stomach
and is abolished by tetrodotoxin, suggesting the ENS is also capable of inducing reflex
relaxation in the absence of extrinsic innervation 28, 75. In vivo, in the presence of a
longstanding vagotomy, reflex gastric relaxation is preserved in animals and humans 28, 30,
77. In rats studied in vivo, surgical vagotomy resulted in a reduced postprandial
accommodation response, but the gastric accommodation reflex was fully restored four
weeks after vagotomy. This preserved response in chronically vagotomized rats was
significantly reduced by tetrodotoxin, but was not affected by guanethidine or
splanchnicotomy, suggesting it is a neurally-mediated response that does not involve
sympathetic pathways 28.
In humans, Azpiroz and Malagelada showed that reflex gastric relaxation in
response to distension was not significantly different in vagotomized patients with
gastroparesis compared to healthy controls41. In contrast, they showed differences in the
phasic volume events in the proximal stomach, which were abolished in vagotomized
patients 104. Others have also documented a normal gastric relaxation in response to a
meal in longstanding vagotomized patients 77. Moreover, in a prospective study in which
the gastric relaxation response was assessed preoperatively and six weeks and one year
after proximal gastric vagotomy, the accommodation reflex was impaired early after
surgery but recovered partially after one year 30. The diabetic patients participating in our
study had a normal gastric volume response to a meal, despite evidence of vagal
neuropathy. This suggests the existence of a non-vagal control of the gastric volume that
allows the postprandial volume increase in the presence of vagal dysfunction. This is
consistent with other reports showing similar gastric accommodation to distension in
vagotomized subjects compared to healthy controls 104 and preserved gastric
accommodation in response to a meal in vagotomized animals 28, 78 and after surgical
vagotomy in humans 30, 77. These data from several studies are consistent, and yet, at first
glance they appear to contradict conventional wisdom. Thus, several studies in the
literature have suggested gastric compliance and accommodation are reduced in patients
with diabetic vagal neuropathy 50, 51, 79. Samson et al. reported 51 decreased gastric
compliance in diabetic patients with autonomic dysfunction plus dyspeptic symptoms but
Delgado-Aros S. - 24 -
accommodation to a meal was not tested. In a later study 50, the same group showed that a
group of diabetic patients had decreased postprandial gastric accommodation response;
however, careful scrutiny of individual data shows that 6 out of 9 diabetic patients with
vagal neuropathy presented a normal accommodation response. Undeland et al.79 also
reported decreased gastric volume response to feeding in diabetic patients with decreased
vagal tone. However, they compared tertiary referral diabetic patients to non-matched
healthy medical students in contrast to our study that recruited community diabetes
without dyspepsia. In all these reports, the accommodation responses of diabetic and
healthy subjects overlap considerably, and the inclusion of referred diabetic patients with
symptoms may confound assessment of the effects of diabetic neuropathy on gastric
accommodation. Dyspeptic symptoms in diabetic patients may be related to factors other
than vagal neuropathy and, symptoms may conceivably induce changes in gastric motor
function.
To avoid this potential confounder, we studied diabetic patients from the
community with evidence of vagal impairment and excluded patients with
gastrointestinal symptoms. We considered these selection criteria were essential to
answer the question whether the gastric volume response to a meal is impaired in the
presence of chronic vagal neuropathy. Our studies lead to the hypothesis that there is
adaptation of the gastric accommodation response to feeding, and this needs to be
assessed in a larger study in which the mechanisms potentially involved in this adaptation
can be explored. However, it is important to point out that our observation is backed by a
large volume of data from experimental animal and human studies 28, 30, 77, 78, 104.
Given the evidence that hyperglycemia may alter gastric tone 105-107, we
performed all the studies under euglycemic conditions to avoid this potential confounder.
Hence, we believe that the normal gastric volume response to the meal in vagotomized
diabetic patients is not the result of differences in postprandial glycemia. We postulate
that, in the presence of vagal dysfunction, the postprandial gastric volume response
occurs through a non-vagal entero-enteric reflex. Our studies do not determine whether
this is a peripheral reflex arc via prevertebral ganglia or a local enteric reflex. Moreover,
we cannot exclude a potential contribution of a hormonal mechanism in the adaptation of
the stomach volume increase after the meal.
Delgado-Aros S. - 25 -
In summary, by evaluating its effects in patients with vagal neuropathy, we have
shown that the GLP-1-induced increase in gastric volume in humans is likely to be
mediated through the vagus. Further studies will be needed to explore whether GLP-1
increases gastric volume by inhibition of vagal cholinergic innervation or by activating
vagal NANC or adrenergic inhibitory pathways. We have also shown that chronic vagal
denervation secondary to diabetes does not necessarily impair gastric accommodation, as
had been suggested by some studies 50, 79, 108. Our data confirm prior experimental animal
studies 28, 78 that suggested the potential of non-vagal pathways to induce gastric reflex
responses to feeding, including the accommodation response.
Delgado-Aros S. - 26 -
3. Figures
Fig. 1. 99mTc-SPECT technique to measure gastric volume. Ten minutes after intravenous injection of 99mTc-sodium pertechnetate to allow gastric mucosal uptake of the isotope, dynamic tomographic acquisition was performed with the single photon emission computed tomography (SPECT) camera. Tomographic images were processed to obtain a 3-dimensional stomach and its volume.
Delgado-Aros S. - 27 -
Fig. 2. Protocol 1. Gastric volumes and plasma levels of glucose and human pancreatic polypeptide (HPP) were obtained at baseline, before glucagon-like peptide-1 (GLP-1) or placebo infusion, and during fasting and the postprandial period, while GLP-1 or placebo infusion was ongoing.
Fig. 3. Protocol 2. Measurement of maximum tolerable volume (MTV), gastric emptying, and postprandial symptoms. Participants drank Ensure at a constant rate until maximum satiety was reached (i.e., MTV). The second glass of Ensure ingested was radiolabeled with 111In-diethylenetriaminepentaacetic acid (DTPA) to measure gastric emptying. Thirty minutes after the drink test was finished, postprandial symptoms were assessed using a visual analog scale (VAS).
Delgado-Aros S. - 28 -
Fig. 4. Examples of gastric volumes obtained at baseline, during fasting, and during postprandial periods from 2 participants in the study, 1 treated with placebo and the other with GLP-1. Note the visibly larger volume of the stomach with GLP-1 infusion.
Fig. 5. Individual values for the MTV ingested in each of the 2 groups.
Delgado-Aros S. - 29 -
Fig. 6. Gastric emptying of a nutrient liquid meal. Gastric emptying curves observed following radiolabeled Ensure ingestion are shown. The primary end point for assessing the gastric emptying effects of GLP-1 and placebo was the proportion emptied at the time the infusion ended, at 30 min. Median proportion emptied in the GLP-1 group was 7% and in the placebo group was 23%. Data shown are median and interquartile ranges. *P < 0.05.
Delgado-Aros S. - 30 -
Fig. 7. Plasma levels of glucose (A) and HPP (B). GLP-1 decreased plasma levels of glucose and inhibited the normal postprandial increase in HPP. Bars are medians; error bars show interquartile ranges. *P < 0.05.
Delgado-Aros S. - 31 -
Fig. 8. Gastric volumes were obtained at baseline, prior to GLP-1 or placebo infusion, and during fasting and the postprandial period, while GLP-1 or placebo infusion was ongoing.
0 10 20 30 40 50 63 7360 80
GLP-1/ Placebo Infusion
MealFasting PostprandialBaseline
Steady StateSPECT Images
I.v 99mTc04-
Delgado-Aros S. - 32 -
Fig. 9. Gastric volume change in response to placebo or GLP-1, during fasting and postprandially, in healthy and diabetic subjects. GLP-1 only increased gastric volumes in healthy individuals. Data shown are median (IQR).
0
100
200
300
400
500
600
700
Fasting Change Postprandial Change
mLPlaceboGLP-1
0
100
200
300
400
500
600
700
Fasting Change Postprandial Change
mLPlaceboGLP-1
Health Diabetes
p=0.005
p=0.009
p=0.5
p=0.3
Delgado-Aros S. - 33 -
Fig. 10. Examples of stomach reconstructions before meal and postprandially under the effect of placebo in healthy (A) and diabetic patients (B). Note the normal increase in gastric volume after the meal in diabetic patients.
(A)
(B)
Delgado-Aros S. - 34 -
Fig. 11. Plasma levels of glucose during both study days in diabetic patients. There were no significant differences at any time point. Data shown are median and interquartile ranges. NS: p>0.05
60
80
100
120
140
160
180
0 50 60 70
minutes
plas
ma
gluc
ose
(mg/
dL)
PlaceboGLP-1
NS NS NS NS
Delgado-Aros S. - 35 -
4. Tables
GLP-1n=13
Placebon=11 P value
Gender (m/f) 5/8 5/6 0.7
Age (years) 33(26-39.5)
33(28-43) 0.5
BMI (Kg/m2) 25(21-26)
28(24-29) 0.4
Baseline HPP (pg/mL) 77(60-104)
99(55-134) 0.8
Baseline Glucose.(mg/dL)
90(82-99)
92(88-95) 0.6
Baseline Gastric Volume
Table 1. Demographic and Baseline Variables.
Values are median ( interquartile range). n= number of subjects in each group.NS: p > 0.05.
239(169-346)
205(176-262) 0.6
115(80-180)
112(82-164) proximal 2/3 (mL)
total (mL)
0.8
Delgado-Aros S. - 36 -
GLP-1n=13
Placebon=11 P value
Baseline Vol. 239(169-346)
205(176-262) 0.6
Fasting Vol. 312(253-365)
225(185-239) 0.002
Table 2. Total Gastric Volumes (mL) and
Fasting Ratio 1.48(1.26-1.60)
1.08(0.90-1.12) 0.003
Postprandial Vol.848
(789-899)651
(602-801) 0.004
Postprandial Ratio 3.53 3.14 0.15 (3.19-4.39) (2.79-3.61)
Values are median ( interquartile range). n= number of subjects in eachgroup. NS: p value > 0.05.
Fasting Difference (61-128) (-21-25) 0.005 80 17
Postprand. Difference (532-671) (401-549) 0.008 608 435
Delgado-Aros S. - 37 -
GLP-1n=13
Placebon=11 P value
Baseline Vol. 115(80-180)
112(82-164) 0.8
Fasting Vol. 147(112-243)
137(97-156) 0.2
1.34 1.07Fasting Ratio (1.04-1.90) (0.86-1.21) 0.5
Table 3. Proximal Gastric Volumes (mL)
Postprandial Vol.629
(581-647)455
(338-485) <0.0001
Postprandial Ratio 5.77 3.72 0.08 (3.42-7.45) (2.84-4.81)
Values are median ( interquartile range). n= number of subjects in eachgroup. NS: p value > 0.05.
Postprand. Difference (400-553) (223-389) 0.0003 461 302
Fasting Difference (8-85) (-23-24) 0.3 43 5
Delgado-Aros S. - 38 -
GLP-1n=13
Placebon=11 P value
1119(874-1546)
1350(1082-1606)
0.16
12.5(6.3-15.8)
10(8-17) 0.7
MTV (mL)
Aggregate Satiety Score(end GLP-1 infusion)
Table 4. Maximum Tolerable Volume and Postprandial Symptoms.
40(8.5-60.5)
35(4-51)
0.4
69(37-79.5)
47(34-69)
0.4
77(65-86.5)
70(67-79)
0.4
4(0-34)
10(4-17) 0.6
Nausea Score
Bloating Score
Fullness Score
Pain Score
Values are median ( interquartile range). n= number of subjects in each group. NS: p value> 0.05.
Aggregate Symptoms Score(30 min.Postprandial)
185(121-250)
169(121-199)
0.5
Delgado-Aros S. - 39 -
Table 5. Demographic Variables
Healthy Placebo Healthy GLP-1 Diabetic
Age (y) 33 (28-43) 33 (26-39) 65 (56-74)
Gender (M/F) 5/6 5/8 5/2
Body Mass Index (Kg/m2) 28 (24-29) 25 (21-26) 26 (24-35)
Diabetes Mellitus Type 1/2 NA NA 5/2
Values are median (interquartile range).
Delgado-Aros S. - 41 -
Table 6. Effect of GLP-1 on Gastric Volume (mL)
DIABETES WITH VAGAL NEUROPATHY
Placebo (n=7) GLP-1 (n=7) P
Baseline Volume 157(139-231) 193(166-258) 0.4
Fasting Volume 208(145-232) 202(158-255) 0.3
Fasting Difference 4(-14 - 50) 5(-3 - 30) 0.5
Fasting Ratio 1.02(0.92-1.32) 1.03(0.99-1.09) 0.5
Postprandial Volume 631(582-814) 677(605-855) 0.1
Postprandial Difference 452(400-493) 469(383-563) 0.3
Postprandial Ratio 3.99(2.73-4.26) 3.18(2.72-4.43) 0.5
HEALTHY CONTROLS
Placebo (n=11) GLP-1 (n=13) P
Baseline Volume 205(176-262) 239(169-346) 0.6
Fasting Volume 225(185-239) 312(253-365) 0.002
Fasting Difference 17(-21 - 25) 80(61-128) 0.005
Fasting Ratio 1.08(0.9-1.12) 1.48(1.26-1.6) 0.003
Postprandial Volume 699(586-774) 855(761-886) 0.01
Postprandial Difference 442(381-533) 615(487-649) 0.009
Postprandial Ratio 3.03(2.72-3.49) 3.42(3.08-4.31) 0.1
Values are median (interquartile range). n = number of subjects in each group.
Delgado-Aros S. - 42 -
Table 7. Effect of GLP-1 on Proximal Gastric Volume (mL)
DIABETES WITH VAGAL NEUROPATHY
Placebo (n=7) GLP-1 (n=7) P
Baseline Volume 71(61-173) 125(105-164) 0.2
Fasting Volume 106(72-177) 125(104-157) 0.3
Fasting Difference 10(-9 - 54) 3(-7 - 26) 0.9
Fasting Ratio 1.15(0.93-1.33) 1.03(0.95-1.12) 0.8
Postprandial Volume 454(406-537) 446(398-565) 0.2
Postprandial Difference 366(294-402) 355(293-409) 0.5
Postprandial Ratio 3.54(2.96-6.69) 3.56(2.84-4.62) 0.7
HEALTHY CONTROLS
Placebo (n=11) GLP-1 (n=13) P
Baseline Volume 112(82-164) 115(80-180) 0.8
Fasting Volume 137(97-156) 147(112-243) 0.3
Fasting Difference 5(-23 - 24) 43(8-85) 0.1
Fasting Ratio 1.07(0.86-1.21) 1.34(1.04-1.9) 0.2
Postprandial Volume 457(330-526) 623(554-640) 0.003
Postprandial Difference 328(184-414) 447(379-546) 0.01
Postprandial Ratio 4.14(3.0-5.15) 5.81(3.19-7.33) 0.2
Values are median (interquartile range). n = number of subjects in each group.
Delgado-Aros S. - 43 -
Table 8. Gastric Volumes (mL) in Healthy Controls and Diabetes with Vagal Neuropathy
Diabetes Placebo (n=7) Healthy Placebo (n=11) P
TOTAL GASTRIC VOLUME
Fasting Volume 208(145-232) 225(185-239) 0.7
Postprandial Volume 631(582-814) 699(586-774) 0.7
Postprandial Difference 452(400-493) 442(381-533) 0.9
Postprandial Ratio 3.99(2.73-4.26) 3.03(2.72-3.49) 0.7
PROXIMAL GASTRIC VOLUME
Fasting Volume 106(72-177) 137(97-156) 0.6
Postprandial Volume 454(406-537) 457(330-526) 0.9
Postprandial Difference 366(294-402) 328(184-414) 0.8
Postprandial Ratio 3.54(2.96-6.69) 4.14(3.0-5.15) 0.9
Values are median (interquartile range). n = number of subjects in each group.
Delgado-Aros S. - 44 -
5. Final Remarks GLP-1 increases gastric volume in healthy humans. Potential mechanism of
action
In these studies we have shown a previously unknown effect of GLP-1 on gastric
motor function, that is an inhibition of gastric wall tone, as expressed by the increase in
gastric volume measured by the SPECT technique. Moreover, we have shown that this
effect is absent in the presence of cardio-vagal neuropathy associated with diabetes. This
suggests that GLP-1 inhibits gastric tone through a mechanism that involves the vagus
nerve.
Based on our data and previous reports, the inhibition of such a wide spectrum of
gastrointestinal functions by GLP-1 would suggest that this peptide is acting through
inhibition of vagal reflexes in response to different stimulus in the gut, most likely at the
level of the afferent pathways, as suggested by others previously 24.
Nevertheless, positive immunoreactivity for GLP-1 and binding of radiolabeled GLP-
1 have been demonstrated at different levels of the brainstem, such as at the NTS, area
postrema, DMVN, the thalamus or hypothalamus 109-112. This suggests that GLP-1 may
act as a modulator or regulator in the gut-brain axis at different levels.
Moreover, our data are consistent with an inhibitory effect of GLP-1 on vagal
cholinergic efferents. In our first study we observed a decrease in pancreatic polypeptide
secretion after meal ingestion in the group receiving GLP-1. The secretion of pancreatic
polypeptide is under vagal cholinergic control during fasting and during the cephalic
phase of the postprandial period 66-69. During the gastrointestinal phase of the
postprandial period, the secretion of this hormone is not only under vagal extrinsic
control, but also influenced by enteropancreatic neural, mostly vagal-cholinergic, and
hormonal reflexes 69, 113, 114. In our study, we observed a median decrease of fasting
pancreatic polypeptide plasma levels of 12mg/dL in the group that received GLP-1,
whereas there was a median decrease of 0.25 pg/mL in the placebo group. This difference
did not reach statistical significance. After the meal, the median increase in pancreatic
polypeptide plasmatic levels in the GLP-1 group was 6.5 pg/mL compared to 119.8
pg/mL. Since only the gastric phase of pancreatic polypeptide secretion was measured
after the meal, and GLP-1 significantly delayed the rate nutrients entered the small
Delgado-Aros S. - 45 -
intestine, we cannot exclude that the difference in the postprandial plasma levels of
pancreatic polypeptide could be due to the difference in the enteric stimuli of pancreatic
polypeptide secretion. Nevertheless, other studies have shown that exogenous GLP-1
decreases pancreatic polypeptide secretion independently of the gastric emptying rate 44.
GLP-1 also inhibits centrally-induced pancreatic and gastric acid secretions 42, effects
that are lost after abdominal vagotomy in animals 23 and humans 22. These data concur
with the hypothesis of an inhibitory effect of GLP-1 on vagal cholinergic efferents.
Inhibition of vagal cholinergic input into the stomach by GLP-1 would induce an
increase in gastric volume (relaxation) in the interdigestive state. This would also add to
the physiological postprandial gastric relaxation through vagal nitrergic activation, and
explain the enhanced volumes observed after the meal during GLP-1 infusion compared
to placebo. (See Introductory Schema)
It is also conceivable that in the NTS, a region where afferent information from the gut
and other organs is integrated, or at the level of DMVN, locally synthesized GLP-1
induces activation of gastric relaxatory reflex by activation of post-synaptic neurons
involved in vagal nitrergic or adrenergic inhibitory pathways. This would induce an
increase in gastric volume (relaxation) during the interdigestive state and enhance
postprandial gastric relaxation, as observed in our studies.
Further studies will be needed to explore whether GLP-1 increases gastric volume
through inhibition of vagal cholinergic post-synaptic neurons or by stimulating post-
synaptic nitrergic or adrenergic inhibitory pathways.
GLP-1 effects on gastric volumes are lost in patients with vagal neuropathy
associated with diabetes
In a group of seven diabetic patients affected by cardiovagal neuropathy, we did not
observe the effect of GLP-1 on gastric volume, which had been documented in healthy
volunteers. The patient group differed from the healthy control group, not only in the
presence of vagal neuropathy, but also in age and the presence of the diabetes itself with
its potential for associated clinical complications. Therefore, one may argue that the lack
of effect of GLP-1 on gastric volume in diabetic patients with vagal neuropathy, may
have been related to differences between the two groups other than the vagal neuropathy.
For instance, the incretin effect of GLP-1 is known to be impaired in diabetic patients,
Delgado-Aros S. - 46 -
presumably due to a reduced responsiveness of the islet β cells to incretins in these
patients 115. Therefore, it is conceivable that in diabetic patients other effects of GLP-1
may be also impaired due to a reduced responsiveness of the GLP-1 receptors,
irrespective of the status of vagal innervation. However, if this is true, we would expect
that effects of GLP-1 on other functions would not be present in diabetic patients with
normal vagal function. On the contrary, GLP-1 effects on gastrointestinal responses, such
as, gastric emptying, appetite or pancreatic secretion, have been shown to be preserved in
diabetic patients with non-insulin dependent diabetes mellitus (NIDDM) 7, 10, 14, 41, 116.
This is strong evidence against the hypothesis that there was a diminished response to
GLP-1 due to the diabetes per se, independent of vagal function.
The diabetic participants in our study were clearly older than the healthy controls.
Aging could have also contributed to the observed differences in the effect of GLP-1 on
gastric volume in the diabetic patients compared to the healthy controls, independently of
the vagal function. Against this argument, it has been shown that effectiveness of GLP-1
is not significantly affected by age 117. Moreover, our diabetic participants are similar in
age to the diabetic samples included in studies mentioned above 7, 10, 14, 116. These studies
showed the preserved effects of GLP-1 on gastric emptying, appetite, or pancreatic
secretion in elderly diabetic patients.
Therefore, we believe our results are more consistent with a lack of response of the
stomach volume to GLP-1 infusion due to vagal impairment. This is also consistent with
the observed lost of GLP-1 effects on gastric acid secretion after vagotomy in non-
diabetic humans 22.
Normal gastric volume response to a meal in patients with vagal neuropathy
associated with diabetes
When testing the effects of GLP-1 on gastric volumes in patients with vagal
neuropathy associated with diabetes, we observed an unexpected finding, that is, that the
gastric volume response after the meal was comparable to that of a healthy control group.
We have already thoroughly discussed the evidence of the literature against and in favor
of this observation (Discussion p31.)
Vagal participation in the control of gastric tone, and hence in gastric relaxation
responses, has been acknowledged for a long time 27, 118-121. However, it is also
Delgado-Aros S. - 47 -
recognized that splanchnic nerves and intrinsic neuronal and humoral mechanisms are
also involved in the control of the tone of the stomach 122, 123. Furthermore, there is
increasing evidence that gastric tone may be adequately controlled in the absence of
extrinsic vagal innervation 28, 104. Studies in animals and humans have shown that gastric
relaxation responses recover with the passage of time after vagal denervation 28, 30, 77, 78.
Others have suggested that the postprandial gastric relaxation response is impaired in
patients after vagotomy 72. In this study from Troncon et al. 72 the gastric volume
responses to imposed pressures and to a standard meal were studied by means of an
intragastric balloon connected to a barostat device. They compared the results in seven
dyspeptic patients that had undergone bilateral truncal vagotomy and pyloroplasty for
peptic ulcer with the results in eleven healthy volunteers. The gastric volume response to
distention was similar in both groups. Overall, they reported decreased gastric volume
response after meal ingestion in the vagotomized dyspeptic patients. Nevertheless, a
careful review of the data in this study shows a normal postprandial gastric relaxation
response in three of the seven vagotomized patients, and an unusual absence of gastric
accommodation after meal ingestion in two of the eleven healthy volunteers. These
unusual observations may have led to erroneous conclusions, and it is relevant that almost
45% (3/7) of vagotomized patients presented normal accommodation.
Previous studies have reported impaired gastric relaxation after meal ingestion in
diabetic patients diagnosed withcardio-vagal neuropathy 79. We have also discussed some
of the concerns regarding the validity of those studies based on variability of gastric
volume responses, inappropriate controls and the presence of concomitant dyspeptic
symptoms in diabetic patients (Discussion p30.).
Since we used an indirect measurement of vagal dysfunction, one could argue that the
normal gastric volume response to a meal observed in our diabetic participants could be
explained by the lack of a real vagal neuropathy in these patients. Certainly, since these
were in vivo studies, rather than pathological studies of the vagus nerve, we cannot
exclude this possibility. Nonetheless, heart rate variability in response to deep breathing
is the gold standard test currently used to diagnose cardio-vagal neuropathy 89-91. It is
considered that gastric vagal function is affected when cardio-vagal impairment is
present, since the vagus is a long nerve and metabolic neuropathies start in distal fields
Delgado-Aros S. - 48 -
before progressing proximally. Thus, demonstration of functional impairment in the
territory innervated by more proximal vagal branches, such as the cardiac nerves, implies
more distal impairment of the nerve (e.g., gastric branches). This has been shown in
several studies performed by pathologists 81-83. When trying to develop a test for an early
diagnosis of vagal neuropathy in diabetic patients, it was found that impaired abdominal
vagal responses (ie pancreatic polypeptide and gastrin responses to sham feeding)
preceded the development of cardio-vagal impairment, and it was always present in
patients with positive cardiovagal neuropathy 84-87. Therefore, a positive test of cardio-
vagal dysfunction implies abdominal vagal dysfunction. Thus, we believe that our
diabetic participants present the features of diabetes autonomic vagal neuropathy, and
that the normal gastric volume response to a meal observed in the presence of vagal
neuropathy adds to the growing evidence of the existence of an adaptive response
preserving postprandial gastric relaxation when the main vago-vagal reflex is impaired.
Delgado-Aros S. - 49 -
6. Summary and Future Studies The presented data provides new information on the effects of GLP-1 on upper
gastrointestinal functions and increases the knowledge of its mechanism of action in
humans. These data also suggests that the gastric accommodation response, a long-
recognized vago-vagal reflex present in animals and humans, is maintained in the
presence of diabetes associated with vagal neuropathy, suggesting that adaptation of other
extrinsic or intrinsic neural pathways compensate for vagal denervation. This is
consistent with previously observed adaptation of other visceral vago-vagal reflexes in
animals and humans.
Further studies are required to evaluate the mechanism of action of GLP-1 on
inhibition of gastric tone. Several plausible hypotheses may be proposed from an
appraisal of the presented results and the literature. Experimental data suggest that
radiolabeled GLP-1 can bind to DMVN neurons. It is possible that GLP-1 could bind and
inhibit cholinergic motor neurons in the DMVN. Inhibition of this pathway would inhibit
gastric tone consistent with an inhibitory effect of GLP-1 on vagal cholinergic efferents.
Secondly, it is also possible that GLP-1 binds to cholinergic motor neurons in the DMVN
that activate nitrergic neurons in the stomach 124, or GLP-1 may bind directly to nitrergic
motor neurons in the DMNV 125, 126. Activation of the last two pathways would also lead
to inhibition of gastric tone 126. Concomitant studies with the nitric oxide synthase
inhibitor L-NMMA and GLP-1 would allow us to test the hypothesis that nitrergic
pathways participate in the inhibition of gastric tone by GLP-1.
A further series of studies in the future should explore whether endogenous GLP-1,
released in the small intestine and colon after meal ingestion, plays a role in the control of
gastric accommodation to a meal. Thus, (9-39)-exendin, an antagonist of the GLP-1
receptor, could be used to test this interesting hypothesis.
Delgado-Aros S. - 50 -
7. Acknowledgments I wish to specially thank my mentor, Dr. Michael Camilleri. Without his critical
advice, help and support, this work would not have been possible.
I wish to thank Dr. Adrian Vella for his valuable help and advice in designing and
setting up the studies using GLP-1 and reviewing the final manuscript.
I thank Benjamin Brinkmann for his assistance with SPECT data analysis, Duane
Burton and George Thomforde for technical support, Cindy Stanislav for her wonderful
secretarial assistance and Dr. Malagelada for his support and review of the final
manuscript.
Delgado-Aros S. - 51 -
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